【作者】 林峰；

【导师】 傅志仁； 丁国善； 王全兴；

【作者基本信息】 第二军医大学 ， 普通外科学， 2008， 博士

【摘要】 肝脏缺血再灌注损伤是肝脏移植中常见的问题之一,缺血再灌注引起的肝脏损伤是一个连续不断的过程,并最终导致肝细胞损伤。这个过程是在肝脏短暂缺氧并再氧合时触发,临床上许多情况下可发生肝脏缺血再灌注,例如肝脏低灌流状态,各种各样的外科手术操作,以及移植中的供体器官切取。实际上肝脏缺血再灌注损伤是抗原成分获取的结果,是影响肝脏移植结果的重要因素,引起10%左右的早期移植后器官功能衰竭,并且急性和慢性排异的发生率较高。此外,由于边缘性供体对于缺血再灌注损伤非常敏感,这加剧了供体器官的短缺。因此减少缺血再灌注损伤能明显增加获得手术成功的病人数量。然而,到目前为止仍没有可以预防缺血再灌注损伤的有效方法。所以,为了保护细胞和器官的功能,需要通过广泛的研究更好的了解肝脏缺血再灌注损伤的机制以及寻找合适的干预方法必要的。青藤碱是从防己科防己属植物青风藤Sinomenium acutum Rehd.et Wils.根茎中分离出的主要活性成分,其结构类似于吗啡,分子式为C₁₉H₂₃NO₄,分子量为329.38,具有镇痛,抗炎等作用,临床主要用于类风湿病的治疗,疗效确切。国内外的研究表明,青藤碱具有良好的抗炎、免疫抑制作用。但是在缺血再灌注损伤中的作用却未见报道。本课题应用“二袖套”法建立大鼠原位肝移植模型,研究青藤碱在肝脏缺血再灌注损伤中的保护作用,并对其作用机制进行探讨。第一部分:青藤碱对大鼠原位肝移植缺血再灌注损伤的保护作用目的:探讨青藤碱对大鼠原位肝移植缺血再灌注损伤的保护作用。方法:应用SD大鼠为模型动物;采用Kamada’s“二袖套法”建立SD→SD大鼠原位肝移植模型,大鼠196只随机分为假手术组、生理盐水对照组、低剂量（40mg/kg）和高剂量青藤碱治疗组（80mg/kg）,将供肝在4℃林格液中保存5h后植入受体,分别观察移植术后1周存活率,检测移植术后2,6,12,24h血清ALT、AST,TUNEL法检测肝细胞凋亡指数（AI）,RT-PCR检测肝脏组织中的TNF-α、IL-β、ICAM-1mRNA的含量,ELISA检测血清IL-2含量,并观察移植肝脏病理形态学的改变。结果:与对照组比较,青藤碱低剂量、高剂量治疗组术后1周存活率显著提高（75%,75%vs12.5%,P＜0.01）,在各个时间点,青藤碱治疗组的ALT、AST水平明显低于生理盐水对照组（P＜0.01）,肝组织中的TNF-α、IL-1β、ICAM-1mRNA表达与对照组相比均显著减少（P＜0.01）,肝细胞凋亡指数下降,在肝移植术后24小时,生理盐水对照组的AI为37.0±4.30,而在青藤碱两治疗组分别为23.8±3.27、18.6±3.03,与对照组相比有显著差异（P＜0.01）。血清IL-2含量降低,肝细胞和肝窦内皮细胞形态也明显改善。结论:青藤碱可以通过抑制Kupffer细胞激活及释放TNF-α、IL-1β等炎症性细胞因子及ICAM-1等粘附分子,抑制肝细胞凋亡,减少细胞免疫相关IL-2的分泌,减轻肝细胞及肝窦内皮细胞的损伤,达到保护肝移植缺血再灌注损伤的效果。第二部分青藤碱对大鼠肝移植缺血再灌注后TLR4、MAPKs和NF-κB表达的影响目的:通过观察不同剂量的青藤碱对大鼠肝移植术后TLR4、MAPKs和NF-κB的影响,探讨青藤碱保护大鼠肝移植缺血再灌注损伤的机制。方法:应用“二袖套法”建立SD→SD大鼠原位肝移植模型,大鼠196只随机分为假手术组、对照组、低剂量（40mg/kg）和高剂量青藤碱治疗组（80mg/kg）,将供肝在4℃林格液中保存5h后植入受体,选择缺血再灌注后大鼠肝功能损伤最严重的术后6小时为观察点。采用Western Blot方法检测大鼠肝脏组织的TLR4、磷酸化JNK、ERK、p38 MAPK和NF-κB蛋白表达的变化情况。结果:在假手术组TLR4呈低表达,生理盐水对照组TLR4呈高表达,青藤碱显著降低了肝组织TLR4的表达（P＜0.01）。磷酸化JNK、ERK在各组的表达比较无明显差异。磷酸化p38 MAPK和NF-κB在假手术组呈低表达,在生理盐水对照组高表达,而青藤碱明显抑制了磷酸化p38 MAPK和NF-κB的表达（P＜0.01）。结论:青藤碱保护大鼠肝移植缺血再灌注损伤的机制与抑制TLR4介导的天然免疫反应有关,通过抑制p38MAPK信号通路和NF-κB通路,减少下游TNF-α、IL-1β和ICAM-1等炎症细胞因子的分泌。第三部分青藤碱对肝脏缺血再灌注后抗原提呈细胞的影响目的:通过观察不同剂量的青藤碱对大鼠肝移植缺血再灌注后肝脏来源的DC成熟和功能的影响,深入了解青藤碱保护缺血再灌注损伤作用的机制。方法:应用“二袖套法”建立SD→SD大鼠原位肝移植模型,大鼠48只随机分为生理盐水对照组、低剂量（40mg/kg）和高剂量青藤碱治疗组（80mg/kg）,将供肝在4℃林格液中保存2h后植入受体,术后第三天切取肝脏,分离纯化肝脏DC,观察青藤碱对其成熟和功能的影响。以荧光抗体标记和流式细胞仪对DC进行表型分析OX62和MHC-Ⅱ、CD86等分子。FACS检测移植后肝脏DC对荧光标记的卵清蛋白（OVA-FITC）能力,分析青藤碱对肝脏DC内吞功能的影响。收集获取的DC,用RT-PCR方法测定其中具有免疫调节作用的细胞因子IL-12,IL-1,TNF-αmRNA的表达水平,Western Blot方法检测各组肝脏DC表达的TLR4。收集各处理组肝脏DC以³H-TdR掺入法进行DC刺激同种异基因T细胞增殖功能（MLR）的检测。结果:FACS分析显示,青藤碱处理组DC呈现不成熟DC的表型,DC表面MHC-Ⅱ类分子和CD86等分子表达均显著下降,而对照组则表现为成熟DC表型,高表达MHC-Ⅱ类分子和CD86分子。内吞功能试验发现青藤碱处理组平均荧光强度较对照组的明显增强,表明了青藤碱对肝脏DC向成熟DC转化具有抑制作用。提示青藤碱可显著降低缺血再灌注后肝脏DC的抗原提呈功能。对照组肝脏DC表达IL-12,IL-1,TNF-αmRNA等水平明显增高,TLR4蛋白的表达明显升高,而青藤碱处理组可不同程度抑制这种效应（P＜0.01）。DC刺激同种T细胞增殖功能（MLR）的检测发现青藤碱处理后肝脏DC的混合淋巴细胞反应明显减弱,提示青藤碱能明显抑制DC向T细胞提呈抗原的能力。结论:青藤碱对大鼠缺血再灌注后肝脏DC的成熟和功能具有抑制作用,青藤碱对肝移植缺血再灌注损伤的保护作用可能通过期其抑制DC成熟和功能,抑制缺血再灌注后T细胞的免疫应答有关。更多还原

【Abstract】 Effect of Sinomenine on ischemia-reperfusion injury in orthotopic rat liver transplantationThe damage to the liver caused by ischemia and reperfusion(I/R) represents a continuum of processes that culminate in hepatocellular injury. These processes are triggered when liver is transiently deprived of oxygen and reoxygenated, and can occur in a number of clinical settings, such as those associated with low flow states, diverse surgical procedures, or during the organ procurement for transplantation. In fact, I/R injury to the liver, an antigenindependent component of "harvesting" insult, represents an important problem affecting transplantation outcome. It causes up to 10% of early organ failure, and can lead to the higher incidence of both acute and chronic rejection. Moreover, I/R injury contributes to the acute shortage of livers available for transplantation because of the higher susceptibility of marginal livers to the ischemic insult. Indeed, minimizing the adverse effects of I/R injury could significantly increase the number of patients that may successfully undergo liver transplantation. However, at present there is no treatment available to prevent hepatic I/R injury. As intervention on more than one level is most likely needed to allow the recovery of cellular and organ function, extensive research efforts to better understand the mechanisms of hepatic I/R injury are warranted.The medicinal plant Sinomenium acutum Rehd.et Wils. has been used in China for more than 2000 years, for the treatment of various diseases. The main active constituent has been identified as the alkaloid Ssinomenine(7, 8-didehydro-4-hydroxy-3, 7-dimethoxy-17-methyl-9α, 13α, 14α-morphinan-6-one). Sinomenine is in clinical use in China and Japan due to its analgetic andanti-inflammatory activities. Its anti-rheumatic properties have been proved inclinical studies.Based on the conclusions mentioned above, in this study, we observe the effect of different dosage of Sinomenine on ischemia and reperfusion injury during orthotopic liver transplantation of rat and try to explore the possible mechanisms.PARTⅠProtective effects of Sinomenine on ischemia-reperfusioninjury during orthotopic liver transplantation in ratsObjective: To investigate the protective effect of Sinomenine on the cold ischemia and reperfusion(I/R) injury during orthotopic liver transplantation (OLT). Methods: We used syngeneic SD as animal model, OLT were performed in normal rats based on Kamada’s two-cuff technique. 196 syngeneic SD rats were randomly divided into sham operation, NS control and two Sinomenine groups. The rats in sinomenine groups were treated with low(40mg/kg) and high dose(80mg/kg) of sinomenine respectively. After the donor liver was preserved in Ringer’s(LR) solution for 5 h, the orthotopic implantation was performed. The serum and tissue samples of 2, 6, 12 and 24 h were collected for analysis after reperfusion of the portal vein, and the one-week survival rate was observed in each group. The apoptosis index(AI) of liver after OLT was detected by TUNEL; The level of TNF-α、IL-1βand ICAM-1mRNA in rat’s liver were detected by RT-PCR; The level of IL-2 in serum was detected by ELISA. Results: In comparison with those in the NS control group, the levels of ALT and AST decreased significantly in low and high dose treatment groups at different time point post-transplantation, and their one-week survival rates also increased markedly (75%, 75% vs 12.5%, P<0.01). The AI was decreased markedly in Sinomenine treatment groups. At the 24h after liver transplantation, the AI of saline control group was 37.0±4.30, and in two Sinomenine treatment groups, the AI wre 23.8±3.27、18.6±3.03, respectively. Compared with saline control group, there was significant difference(P<0.01). Sinomenine treatment decreased the expression of tumor necrosis factor-a(TNF-α)、interleukin-1β(IL-1β) and intercellular adhesion molecule-1(ICAM-1) in rat’s livers, The elevation of serum IL-2 was suppressed by Sinomenine and Sinomenine treatment markedly ameliorated the focal necrosis of hepatocytes. Conclusion: Sinomenine can inhibit hepatocyte apoptosis by decreasing the discharge of TNF-α、IL-1βand ICAM-1, and decrease secretion of IL-2 correlating with T cell-mediated immunity. Sinomenine can prevent hepatic cells and SEC from cold ischemia and reperfusion injury during orthotopic liver transplantation in rats.PARTⅡEffect of Sinomenine on TLR4、MAPKs and NF-κB inrat’s liver after ischemia-reperfusionObjective: To investigate the effect of Sinomenine on TLR4、MAPKs and NF-κBafter ischemia and reperfusion injury during orthotopic liver transplantation (OLT) and the mechanism of Sinomenine’s protection of ischemia and reperfusion injury.Methods: We used syngeneic SD as animal model, OLT were performed in normal rats based on Kamada’s two-cuff technique. 196 syngeneic SD rats were randomly divided into sham operation, NS control and two Sinomenine groups. The rats in Sinomenine groups were treated with low(40mg/kg) and high dose(80mg/kg) respectively. After the donor liver was preserved in Ringer’s(LR) solution for 5 h, the orthotopic implantation was performed. We selected the sixth hour after ischemia and reperfusion injury as our observation points when the rat’s liver function was the worst. The protein expression of TLR4、phosphorylated JNK、ERK、p38 MAPK and NF-kB were detected by Western Blot. Results: In the sham operation group, the TLR4 was low expression. High expression of TLR4 in saline control group, sinomenine decreased significantly the expression of TLR4 in liver tissue (P<0.01).The expression of phosphorylated JNK and ERK were no difference between four group, But the expression of phosphorylated p38 MAPK and NF-κB were low in sham operation group. The expression of phosphorylated p38 MAPK and NF-kB in saline control group were high, Sinomenine decreased significantly the expression of phosphorylated p38 MAPK and NF-kB in liver tissue (P<0.01).Conclusion: The mechanisms of sinomenine’s protection of ischemia-reperfusion injury during liver transplantation in rats was related with its inhibition of innate immune responses mediated by TLR4. Sinomenine reduced the secretion of inflammatory cytokines such as TNF-α、IL-1βand ICAM-1 by inhibiting p38 MAPK and NF-κB signaling pathway. PARTⅢEffect of Sinomenine on the liver APC afterischemia-reperfusionObjective: To understand the Sinomenine’s protective mechanism inischemia-reperfusion injury preferably, we investigated the effects of Sinomenine on maturation of liver derived dendritic cells (DC). Methods: We used syngeneic SD as animal model, OLT were performed in normal rats based on Kamada’s two-cuff technique. 48 syngeneic SD rats were randomly divided into NS control and two Sinomenine groups. The rats in Sinomenine groups were treated with low(40mg/kg) and high dose(80mg/kg), respectively. After the donor liver was preserved in Ringer’s(LR) solution for 2h, the orthotopic implantation was performed. Three days after operation, We Reseced the liver, separated and purificated liver DC, and observated the effects of Sinomenine on the maturion and function of DC. With fluorescent antibody markers and FACS, we analyzed the DC phenotypic expression of MHC-Ⅱ、OX62 and CD86. To investigate the effect of Sinomenine on the endocytosis of DC, we used FACS to analysis DCs endocytosis of protein antigen with OVA-FITC. Then detected the expressof IL-12, IL-1, TNF-αmRNA of liver DC by RT-PCR, and detected the expression of TLR4 on the liver DC of three groups. We collected the liver DC of three groups, and investigated allogeneic mixed lymphocyte reaction with ~3H-TdR. Results: The DC treated with sinomenine showed immature phenotype, and the liver DC of control group showed matured phenotype which MHC-Ⅱand CD86 were high expression. In Sinomenine treatment groups, the average intensity of fluorescence was stronger than that of the control group, and that showed the Sinomenine have the ability of inhibition on conversion of DC from immaturity to maturity. The test of endosome function found that Sinomenine can significantly decrease the antigen presenting function of liver DC. In addition, the liver DC treated with Sinomenine showed the expression of TLR4 and the IL-12、IL-1、TNF-αmRNA were low. At the same time, we found that the Sinomenine can significantly inhibit the liver DC mixed lymphocyte reaction result from MLR. Conclusion: Sinomenine can inhibit the maturity and function of liver DC. The effects of Sinomenine on ischemia-reperfusion injury during orthotopic liver transplantation in rats may be related to inhibition of the immune response mediated by T cell through inhibiting the maturation and function of liver DC.更多还原